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PubMedDate not supplied

Effect of tyrosine kinase inhibitors on tyrosine phosphorylation and motility parameters in human sperm.

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37 degrees C in 5% CO(2), with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. This inhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

Amino Acid Sequence↗
PubMed1981

Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase: relationship to catecholamine metabolism.

Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of alpha-methyl-p-tyrosine (AMT) methyl ester HCl (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.

Animals↗
PubMed1991

The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.

Aluminum↗
PubMed1990

Activation of tyrosine phosphorylation in human T cells via the CD2 pathway. Regulation by the CD45 tyrosine phosphatase.

In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.

Antigens, CD↗
PubMed1994

Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.

Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation.

Amino Acid Sequence↗
PubMed2004

Metformin (Glucophage) inhibits tyrosine phosphatase activity to stimulate the insulin receptor tyrosine kinase.

Metformin is a commonly used anti-diabetic but whether its mechanism involves action on the insulin receptor or on downstream events is still controversial. With a time course that was slow compared with insulin action, metformin increased tyrosine phosphorylation of the regulatory domain of the insulin receptor (specifically, tyrosine residues 1150 and 1151). In a direct action, therapeutic levels of metformin stimulated the tyrosine kinase activity of the soluble intracellular portion of the beta subunit of the human insulin receptor toward a substrate derived from the insulin receptor regulatory domain. However, metformin did not alter the order of substrate phosphorylation by the insulin receptor kinase. Using a Xenopus oocyte preparation, we simultaneously recorded tyrosine kinase and phosphatase activities that regulate the insulin receptor by measuring the tyrosine phosphorylation and dephosphorylation of peptides derived from the regulatory domain of the human insulin receptor. In an indirect stimulation of the insulin receptor, metformin inhibited endogenous tyrosine phosphatases and purified human protein tyrosine phosphatase 1B that dephosphorylate and inhibit the insulin receptor kinase. Thus, there was evidence that metformin acted directly upon the insulin receptor and indirectly through inhibition of tyrosine phosphatases.

Animals↗
PubMed1996

Activation of protein-tyrosine phosphatase SH-PTP2 by a tyrosine-based activation motif of a novel brain molecule.

BIT (a brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (tyrosine-based activation motifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in lambdagt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.

Amino Acid Sequence↗
PubMed2004

A cyclic adenosine 3',5'-monophosphate-induced tyrosine phosphorylation of Syk protein tyrosine kinase in the flagella of boar spermatozoa.

A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5 degrees C for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the anti-phospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+-independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa.

Animals↗
PubMed2000

Tyrosine nitration of c-SRC tyrosine kinase in human pancreatic ductal adenocarcinoma.

During pancreatic tumorigenesis, the equilibrium between cell survival and cell death is altered, allowing aggressive neoplasia and resistance to radiation and chemotherapy. Local oxidative stress is one mechanism regulating programmed cell death and growth and may contribute to both tumor progression and suppression. Our recent in situ immunohistochemical studies demonstrated that levels of total nitrotyrosine, a footprint of the reactive nitrogen species peroxynitrite, are elevated in human pancreatic ductal adenocarcinomas. In this study, quantitative HPLC-EC techniques demonstrated a 21- to 97-fold increase in the overall levels of nitrotyrosine of human pancreatic tumor extracts compared to normal pancreatic extracts. Western blot analysis of human pancreatic tumor extracts showed that tyrosine nitration was restricted to a few specific proteins. Immunoprecipitation coupled with Western analysis identified c-Src tyrosine kinase as a target of both tyrosine nitration and tyrosine phosphorylation. Peroxynitrite treatment of human pancreatic carcinoma cells in vitro resulted in increased tyrosine nitration and tyrosine phosphorylation of c-Src kinase, increased (>2-fold) c-Src kinase activity, and increased association between c-Src kinase and its downstream substrate cortactin. Collectively, these observations suggest that peroxynitrite-mediated tyrosine nitration and tyrosine phosphorylation of c-Src kinase may lead to enhanced tyrosine kinase signaling observed during pancreatic ductal adenocarcinoma growth and metastasis.

Adenocarcinoma↗
PubMed1980

Regional tyrosine levels in rat brain after tyrosine administration.

Endogenous tyrosine concentrations varied two-fold among various rat brain regions, tending to be highest in brain stem structures. Administration of L-tyrosine (100 mg/kg) increased tyrosine concentrations in all brain areas; high relative increases were observed in areas with low initial tyrosine concentrations and vice versa, resulting in a more uniform distribution of tyrosine in the brain. Largest relative increases were observed in cortex and hippocampus. Tyrosine concentrations in all areas reached maximal levels 1 hour after tyrosine was given and declined gradually over the next 3 hours. The results suggest that tyrosine's effects on catecholamine synthesis and release might be amplified in cortex and hippocampus, where highest relative increases in tyrosine concentrations were observed.

Animals↗
PubMed1995

Tubulin-tyrosine ligase catalyzes covalent binding of 3-fluoro-tyrosine to tubulin: kinetic and [19F]NMR studies.

The use of 3-fluoro-tyrosine as an alternative substrate for the enzyme tubulin:tyrosine ligase which catalyzes the incorporation of tyrosine into the alpha-tubulin subunit was investigated. The incorporation of tyrosine into tubulin was inhibited competitively by 3-fluoro-tyrosine with an apparent Ki of approximately 25 microM. The affinity for this analog was similar to that of tyrosine, confirming that the hydrogen at position 3 of the aromatic ring is not essential for the reaction catalyzed by TTLase. The incorporation of 3-fluoro-tyrosine into the C-terminus of the alpha-tubulin subunit was demonstrated through [19F]NMR spectroscopy. The 3-fluoro-tyrosine signal at -58.6 ppm (trifluoroacetic acid as external standard), with a bandwidth of 24.7 Hz presented a chemical shift of 0.75 ppm upfield and an enlargement in the bandwidth (30.5 Hz) when incorporated into tubulin. These results strongly suggest that this amino acid is exposed to the solvent in tubulin. Tubulin covalently labeled with 3-fluoro-tyrosine was competent to polymerize into microtubules. The use of fluorinated tubulin in [19F]NMR spectroscopy for studying questions concerning protein conformation and interactions will be discussed.

Animals↗
PubMed2000

Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation.

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxylase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in tyrosine hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine hydroxylase identified three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in tyrosine hydroxylase at the position corresponding to position 379 of phenylalanine hydroxylase is critical for dihydroxyphenylalanine formation. The relative specificity of tyrosine hydroxylase for phenylalanine versus tyrosine, as measured by the (V/K(phe))/(V/K(tyr)) value, increased by 80000-fold in the D425V enzyme. However, mutation of the corresponding valine 379 of phenylalanine hydroxylase to aspartate was not sufficient to allow phenylalanine hydroxylase to form dihydroxyphenylalanine at rates comparable to that of tyrosine hydroxylase. The double mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, showing a 3000-fold decrease in the (V/K(phe))/(V/K(tyr)) value.

Aspartic Acid↗
PubMed1996

PDGF stimulates tyrosine phosphorylation of JAK 1 protein tyrosine kinase in human mesangial cells.

Platelet-derived growth factor (PDGF) exerts multiple effects in glomerular mesangial cells, including transcription of genes that mediate its biological activity. We have partially characterized PDGF-mediated early mitogenic signal transduction pathways that include activation of protein kinase C alpha and phosphatidylinositol 3 kinase. However, the precise mechanism of PDGF-induced gene transcription is not yet clear. A family of cytoplasmic transcription factors referred to as signal transducers and activators of transcription (STAT) has recently been identified. This group of transcription factors is activated by different cytokines via tyrosine phosphorylation. We studied the effect of PDGF on STATs in human mesangial cells. Using a gel retardation assay, nuclear and cytoplasmic extracts from PDGF-stimulated mesangial cells contained protein factors that bind to a DNA sequence representing the sis-inducible element (SIE) present in the c-fos gene promoter. These protein factors also bind to the enhancer element present in interferon-gamma responsive genes, suggesting the involvement of STAT proteins. The addition of monoclonal antibody that recognizes STAT 1 results in "supershift" of the DNA-protein complex stimulated by PDGF indicating the presence of STAT 1. Immunoblotting experiments with a monoclonal STAT 1 antibody revealed the presence of STAT1 alpha and STAT1 beta in mesangial cells. Since certain cytokines activate STATs via tyrosine phosphorylation mediated by JAK family of tyrosine kinases, we studied the effect of PDGF on JAK kinases. Antiphosphotyrosine immunoblotting of JAK 1 immunoprecipitates from PDGF-stimulated mesangial cell lysate showed increased tyrosine phosphorylation of this tyrosine kinase. In vitro immune complex kinase assay of JAK 1 immunoprecipitates from PDGF-stimulated mesangial cell lysate revealed activation of this tyrosine kinase. Taken together, these data demonstrate that PDGF activates the transcription factor STAT 1 in mesangial cells. The data also provide the first evidence that PDGF stimulates tyrosine phosphorylation of JAK 1, the cytoplasmic tyrosine kinase stimulated by many other cytokines to activate transcription via STATs. These observations indicate that JAK 1 is a downstream tyrosine kinase in PDGF receptor signaling and is a candidate for activation of STAT 1.

Binding Sites↗
PubMed1995

Tyrosine phosphorylation in a model of ischemia using the rat hippocampal slice: specific, long-term decrease in the tyrosine phosphorylation of the postsynaptic glycoprotein PSD-GP180.

The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M(r) 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M(r) 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro "ischemia" was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N2 instead of O2 and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca2+ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca2+ was omitted from the incubation media.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

Animals↗
PubMed1978

L-alanyl-L-tyrosine as a tyrosine source during intravenous nutrition of the rat.

Tyrosine is considered an essential amino acid for some premature infants, but its limited solubility prevents addition to intravenous solutions in adequate amounts. Tyrosine peptides with solubilities greater than tyrosine were synthesized to evaluate potential for intravenous use. Intraperitoneal injection of L-alanyl-L-[U-14C]tyrosine (0.5 mmoles/kg) into adult rats resulted in a rapid labeling of tissue pools and production of 14C-CO2. When the peptide was infused as part of an intravenous nutrition solution (0.5 mmoles/kg/24 hours) into adult rats, plasma and tissue tyrosine pools were rapidly labeled. The radioactivity distribution after 24 hours of infusion was: 41% CO2, 13.4% muscle, 7.7% urine, 7.1% liver, 5.5% intestine, 1.4% kidney, with the remainder in other carcass organs. No unhydrolyzed peptide was found in tissue homogenates. Between 9% and 17% of the radioactivity present in tissue was free tyrosine, with the remainder in tissue protein. Tyrosine accounted for more than 96% of this protein bound radioactivity. The data indicate good utilization of alanyl-tyrosine as a tyrosine source when administered intravenously.

Amino Acids↗
PubMed1982

CD and 1H-n.m.r. studies on the side-chain conformation of tyrosine derivatives and tyrosine residues in di- and tripeptides.

Tyrosine, tyrosine peptides and derivatives, in total 11 species, were selected as models for the study of optical properties (1Lb band of phenolic group) and side-chain arrangement (rotamers around C alpha-C beta bond) of tyrosine as a function of chemical structure and pH effects. Circular dichroism spectra between 240 and 320 nm and n.m.r. spectra were recorded for the different ionization states. Results are discussed in terms of charge effects from N- and C-terminal groups and local conformation influence on 1Lb band of the phenolic chromophore and on distribution of rotamer populations in side-chain of tyrosine. Fractions of rotamer populations were estimated from alpha-beta proton-proton coupling constants and, in the cases of tyrosine and N-acetyl-tyrosine, from 15N-beta nitrogen-proton coupling constants, which allow the stereospecific assignment of the beta and beta' protons. The rotamer populations of tyrosine, averaged from all the data of the samples In solution, were then compared with their statistical distribution in th solid state. Interestingly, agreement is excellent when we refer to crystal of tyrosine, tyrosine derivatives or small peptides (31 samples) and poor in the case of proteins. This leads to a discussion on both the validity of using statistical distributions of rotamers in proteins as reference for rotamer preferences inside small peptides in solution and the choice of the appropriate Jg and Jt values in Pachler's approach. The possible existence of a correlation between ellipticity and rotamer populations for such samples is examined.

Circular Dichroism↗
PubMed1996

Role of the juxtamembrane tyrosine in insulin receptor-mediated tyrosine phosphorylation of p60 endogenous substrates.

Prior studies have demonstrated that a juxtamembrane tyrosine (tyrosine 972) in the insulin receptor is required for the receptor to elicit various biological responses and to stimulate the tyrosine phosphorylation of two endogenous substrates, the insulin receptor substrate-1 and the adaptor protein called Shc. In the present studies the role of this tyrosine was examined in the insulin-stimulated tyrosine phosphorylation of a group of 60-kDa endogenous proteins. These include a 60-kDa protein which, when phosphorylated, becomes associated with the GTPase activating protein of Ras, a distinct 60-kDa protein that associates with either the phosphatidylinositol 3-kinase or the tyrosine phosphatase Syp, as well as a 58/53-kDa protein that is tyrosine phosphorylated in response to insulin but has no known associated protein. In each case, a mutant insulin receptor in which tyrosine 972 has been changed to phenylalanine was found to be defective in its ability to phosphorylate these three endogenous substrates, although the mutant receptor exhibited the same level of insulin-stimulated autophosphorylation as the wild type receptor. These results further demonstrate the critical role that the juxtamembrane tyrosine 972 plays in downstream signaling by the insulin receptor.

Animals↗